In Mohs micrographic surgery, tissue having a tumor, typically a carcinoma on the skin of the head or neck, is excised from a patient under microscopic guidance. The excised tissue specimen, often called a biopsy, is horizontally sliced to provide thin tissue sections that are then histologically prepared on slides. The slides are reviewed under a microscope to determine whether the tumor is fully contained in the excised tissue. This is indicated by the absence of the tumor in the edges or margins of the excised tissue. If the tumor is not fully contained in the excised tissue, additional tissue from the patient is excised and the procedure is repeated until all tissue sections taken indicate the tumor has been removed from the patient. Mohs surgery permits removal of a tumor with maximum preservation of normal surrounding tissue. Mohs surgery is described in the book entitled MOHS SURGERY FUNDAMENTALS AND TECHNIQUES (Kenneth G. Gross, M.D. et al. eds., 1999).
To prepare each tissue specimen in Mohs surgery, multiple sections or slices are manually made with a microtome, where each section is planar and parallel to each other. Often the tissue specimen is first frozen to make the tissue easier to manipulate and cut by the microtome. However, since numerous sections must be made from each tissue specimen and then histologically prepared on slides, this procedure is both tedious and time consuming.
U.S. Pat. No. 4,752,347 provides a method and apparatus for preparing a tissue specimen for sectioning for Mohs surgery. The patent describes placing an excised tissue specimen on a platform, applying a flexible plastic membrane over the tissue specimen, and evacuating the area between the membrane and the tissue specimen. This retracts the membrane onto the platform and pushes the edges of the tissue specimen into a planar orientation parallel to the platform. While under the pressure of the membrane, the tissue sections may be manipulated by an operator through the membrane until the desired orientation is obtained. The edges of the tissue specimen are thus oriented to flatten the edges of the specimen down. The specimen is then frozen, peeled away from the platform, and sectioned by a microtome. Since the edges of the specimen are oriented planar when sectioned by the microtome, a single section can be made having the edges of interest in Mohs surgery. This procedure is adequate for obtaining a section which can be placed on a slide for review under a microscope, but is not useful with optical imaging techniques, such as provided by confocal microscopes, which can examine a surgically exposed tissue specimen without the need for traditional microtome sectioning or slide preparation.
Confocal microscopes optically section tissue to produce microscopic images of tissue sections without requiring histological preparation of the tissue on slides (i.e., slicing, slide mounting, and staining). An example of a confocal microscope is the VivaScope® manufactured by Caliber Imaging Diagnostics, Inc. (formally Lucid Inc.) of Henrietta, N.Y. Other examples of confocal microscopes are described in U.S. Pat. Nos. 5,788,639, 5,880,880, and 7,394,592, and in articles by Milind Rajadhyaksha et al., “In vivo Confocal Scanning Laser Microscopy of Human Skin: Melanin provides strong contrast,” The Journal of Investigative Dermatology, Volume 104, No. 6, June 1995, and Milind Rajadhyaksha and James M. Zavislan, “Confocal laser microscope images tissue in vivo,” Laser Focus World, February 1997, pages 119-127. Further, optically sectioned microscopic images of tissue can be produced by optical coherence tomography or interferometry, such as described in Schmitt et al., “Optical characterization of dense tissues using low-coherence interferometry,” Proc. of SPIE, Volume 1889 (1993), or by a two-photon laser microscope, such as described in U.S. Pat. No. 5,034,613. Raman spectral signatures of molecules can be measured in the skin with optical sectioning microscopy, such as described by Peter J Caspers et al., “In Vivo Confocal Raman Microspectroscopy of the Skin: Noninvasive Determination of Molecular Concentration Profiles”, Journal of Investigative Dermatology (2001) 116, 434-442. Additionally confocal fluorescence microscopes, such as Nikon Instruments AZ-C1 Macro Laser Confocal Imaging System that can image endogenous tissue fluorescence or the fluorescence of exogenous compounds that are applied to the tissue.
One problem with optical sectioning a tissue specimen for Mohs surgery such as by confocal microscope is that the tissue specimen is generally too thick, for example 2-3 mm, to image the edges of the specimen to determine if the specimen contains all of the tumor. Edges refer to areas along the tissue specimen where the cut was made in order to remove the tissue specimen from the patient that may or may not have the margins of the tumor. Often the excised tissue surface is generally convex. It is this convex surface that is needed to be examined to determine if tumor is present in the specimen. Typically, a confocal microscope is limited to producing adequate images of tissue sections at 100-200 microns. Thus, it would be desirable to optically image a tissue specimen in which the edges of the tissue specimen are oriented planar against an optically transparent surface through which the specimen can be optically sectioned.
To overcome this problem, U.S. Pat. No. 6,411,434 describes a cassette having a base member with a rigid optically transparent planar window upon which a tissue specimen is situated, and a pliable plastic membrane locatable over the window and a substantial portion of the base member. With the tissue specimen between the membrane and the window, the edges of the tissue specimen along the sides of the specimen are manually positioned through the membrane so that they lie planar against the window along with the bottom surface of the specimen. The edges may be retained in that position by multiple bonds formed between the membrane and window at points or locations around the tissue specimen. The specimen is imagible by an optical sectioning microscope through the window of the cassette. Although useful, manual positioning needs a skilled technician using a probe to reshape the edges of a thick tissue specimen (e.g., 2-3 mm) to be planar against the planar window surface without puncturing the membrane is a delicate procedure, which if not performed properly can damage the tissue specimen's edges. Thus, it would be desirable to optically image a thick tissue specimen in which the edges needed to be imaged are oriented against an optically transparent window surface through which the specimen can be imaged by an optical sectioning microscope without requiring the need for manually position each of the edges around the specimen so that such edges can be imaged by the microscope.